Project Summary The interactions between mitochondrial metabolism and excitation-contraction coupling play important roles in regulating normal cardiac functions and arrhythmogenesis. In ventricular myocytes, calcium (Ca) is released from the sarcoplasmic reticulum (SR) via the ryanodine receptors (RyRs) and reuptaken back to the SR via the sarco/endoplasmic Ca ATPase (SERCA) pump. During this cycle, mitochondria also uptake Ca and then release it, serving as cytosolic Ca buffers. A ventricular myocyte contains ~7,000 mitochondria and >20,000 Ca release units (CRUs) which are intermingled in space, forming a complex coupling network with local interactions that can cause spatiotemporal subcellular dynamics (e.g. waves and oscillations) of Ca cycling and metabolism. Under the normal condition, a low level of free Ca is needed in the mitochondria to regulate ATP production accompanied with a small amount of reactive oxygen species (ROS) generation by the electron transport chain. Under metabolic stress, mitochondria depolarize, which affect Ca cycling and action potential (AP) dynamics, and induce arrhythmias via different pathways and feedback loops, including: 1) release of a large amount of Ca from the mitochondria to directly affect Ca cycling dynamics and signaling; 2) generation of a large amount of ROS increases ryanodine receptor (RyR) open probability and alters SERCA activity via activation of CaMKII and PKA signaling or redox regulation; 3) lowering ATP production; and 4) reducing gap junction coupling and increasing dispersion of excitability in tissue. In addition, Ca sparks occur randomly due to random L-type Ca channel and RyR openings. Similarly, mitochondrial membrane potential flickering and ROS flashes also occur randomly at the single mitochondrion level. Therefore, to understand the mechanisms of arrhythmias caused by metabolic stress and to identify potential effective therapeutic targets, systems approaches that consider the complex and multiscale regulations are required. This project proposes to combine experiments and multiscale modeling as well as nonlinear dynamics to investigate the spatiotemporal subcellular Ca cycling dynamics and arrhythmogenesis caused by metabolic stress with the following specific Aims: Aim #1. To develop spatially- detailed mouse and rabbit ventricular myocyte models composed of networks of coupled mitochondria and CRUs and use them to investigate the effects of metabolic stress on spatiotemporal subcellular Ca cycling dynamics and cellular AP dynamics; Aim #2. To investigate the effects of metabolic stress on spatiotemporal subcellular Ca cycling dynamics and cellular AP dynamics in ventricular myocytes isolated from different animal models, including wild type, Cyclophilin D knockout, and mitochondrial Ca uniporter knockout mouse hearts and normal rabbit hearts; and Aim #3. To investigate the effects of metabolic stress on ventricular arrhythmias in computer simulation of tissue models and whole-heart experiments of normal and knockout mice and rabbits. Our goal is to understand the general mechanisms of arrhythmias caused by metabolic stress and dissect the differential contributions of the metabolic stress pathways, providing insights into the development of effective therapies.